The overall objective of the proposed research is to define the mechanism of cellular adenosine release with the special regard to this substance's role as a local vasoregulator metabolite. To play such a role, adenosine must first be released from the parenchymal cells into the interstitial (extracellular) space. From there it can relax the smooth muscle in the arterioles, thus affecting tissue blood flow. Two different hypotheses to explain adenosine release from parenchymal cells have been advanced. The traditional view, forwarded by Berne and his colleagues, holds that vasoregulatory adenosine is formed at the cell membrane and released directly to the extracellular space. The newer hypothesis, proposed by Schrader et al., holds that adenosine is formed within the cell and enters the extracellular space by facilitated diffusion down a concentration gradient. In the proposed reasearch, isolated liver and cardiac muscle cells will be studied to facilitate discrimination between intracellular and extracellular adenosine pools. This will allow us to ascertain the location of adenosine production prior to its release. Specifically, we aim to determine the relative importance of membrane-bound 5'-nucleotidase, intracellular adenosine-forming enzymes and facilitated transport in regard to adenosine release from isolated cells. We will also assess the relative importance of various compounds (adenosine 5'-monophosphate, S-adenosylhomocysteine, bound intracellular adenosine) as sources of released adenosine. The elucidation of the mechanisms whereby adenosine is released from parenchymal cells and the identification of the precursors of this potentially vasoactive adenosine are essential to our understanding of local vasoregulatory mechanisms in heart, liver and other tissues. It is also important to understand these features in order to design experiments which are adequate to test adenosine's involvement in local vasoregulation in specific organs under specific circumstances. Finally, knowledge of the cellular details of the adenosine vasoregulatory mechanism will allow rational approaches to developing effective therapeutic measures for the treatment of certain circulatory disorders.